This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. RNA interference techniques have revolutionized our ability to silence genes and hold great promise as a therapeutic approach against a diverse set of diseases. To use this technology optimally, one needs to decipher the molecular basis underlying individual steps in the catalytic cycle that eventually lead to degradation of the targeted message. This project focuses on one critical component of the degradation machinery, the Argonaute (Ago) protein, and addresses its ability to accommodate a nucleic acid template (guide strand) within its scaffold, and defines key features of its alignment for pairing and subsequent nuclease-mediated cleavage of the message. The Ago protein, composed of PAZ- and PIWI-containing modules, as a key catalytic component of the RNA-induced silencing complex (RISC), plays a central role in the RNA interference pathway by mediating sequence-specific cleavage of target messenger RNA (mRNA), including the maturation of small interfering RNA (siRNA) through initial degradation of the passenger strand. Our goal is to determine structures of Ago-nucleic acid complexes during distinct assembly and functional steps of the RNA interference catalytic cycle in order to define the mechanisms underlying guide strand mediated targeting and degradation of mRNA.